周细胞条件培养液对视网膜色素上皮细胞、胶质细胞及巨噬细胞增殖的影响

目的观察体外培养的视网膜周细胞条件培养液对视网膜色素上皮细胞、巨噬细胞及胶质细胞生长的影响。方法MTT法测定不同稀释度的周细胞条件培养液对色素上皮细胞、巨噬细胞及胶质细胞生长的影响。结果随着周细胞条件培养液稀释度的增加,其对上述细胞的抑制生长作用亦越明显。结论周细胞的缺失将导致色素细胞、巨噬细胞及胶质细胞的增殖,细胞间相互作用对于糖尿病视网膜病变的发生和发展是非常重要的。     

胶质细胞源性神经营养因子在小鼠生精恢复过程中的表达和意义

目的:分析并探讨胶质细胞源性神经营养因子(GDNF)在小鼠恢复生精过程中的表达变化,及其与精原干细胞增殖与分化的关系。方法:间隔24 d 2次腹腔注射白消安建立小鼠生精恢复过程的动物模型。根据第二次给药后的不同时间随机分为1、2、3、4、6、8、10周共7组(8只/组),8只正常小鼠作对照组,分别于相应时点取材,进行光镜和电镜的研究。采用半定量逆转录聚合酶链反应(RT-PCR)检测不同时间GDNF mRNA的表达变化;并采用原位杂交技术检测GDNFmRNA表达定位。结果:2次给药后第1-2周,GDNF mRNA的表达明显增强,在第2周达到高峰;在给药后第3-4周明显下降,并在第4周达到低谷;随后几周内表达又逐渐增强,于第10周恢复到正常水平。原位杂交显示,GDNF由sertoli细胞表达。结论:在小鼠生精恢复过程中,GDNF的高水平表达促进精原干细胞自我更新,对于维持生精上皮干细胞数量的稳定具有十分重要的意义。     

碱性成纤维细胞生长因子对新生鼠缺氧缺血性脑损伤胶质纤维酸性蛋白表达的影响

目的探讨外源性碱性成纤维细胞生长因子（bFGF）对新生大鼠缺氧缺血性脑损伤（HIBD）后海马胶质纤维酸性蛋白（GFAP）表达的影响。方法通过结扎并剪断7日龄新生Wistar大鼠右侧颈总动脉，吸入8％氧气和92％氮气2h制备新生鼠HIBD模型，设假手术组、生理盐水对照组、bFGF治疗组。通过免疫组织化学方法和计算机图像分析技术检测3组大鼠不同时点（术后d4、7、10、17、24）海马CAI区GFAP表达强度变化。结果假手术组海马内GFAP阳性细胞数量和染色强度术后d7达高峰：对照组、治疗组GFAP表达较假手术组增多，治疗组增多更明显，术后d10达高峰，GFAP阳性细胞主要分布于海马CAI、CA3区．术后d4、10、17组比较差异有显著性（P均〈0．05）。结论1．新生鼠HIBD后脑缺血易损伤区GFAP表达增加，可能与脑损伤后神经细胞再生有关；2．外源性给予bFGF可增强新生鼠脑缺氧缺血后中枢神经系统GFAP表达，在神经细胞损伤的修复中发挥一定保护作用。     

Astrocytes in the damaged brain: Molecular and cellular insights into their reactive response and healing potential

Long considered merely a trophic and mechanical support to neurons, astrocytes have progressively taken the center stage as their ability to react to acute and chronic neurodegenerative situations became increasingly clear. Reactive astrogliosis starts when trigger molecules produced at the injury site drive astrocytes to leave their quiescent state and become activated. Distinctive morphological and biochemical features characterize this process (cell hypertrophy, upregulation of intermediate filaments, and increased cell proliferation). Moreover, reactive astrocytes migrate towards the injured area to constitute the glial scar, and release factors mediating the tissue inflammatory response and remodeling after lesion. A novel view of astrogliosis derives from the finding that subsets of reactive astrocytes can recapitulate stem cell/progenitor features after damage, fostering the concept of astroglia as a promising target for reparative therapies. But which biochemical/signaling pathways modulate astrogliosis with respect to both the time after injury and the type of damage? Are reactive astrocytes overall beneficial or detrimental for neuroprotection and tissue regeneration? This debate has been animating this research field for several years now, and an integrated view on the results obtained and the possible future perspectives is needed. With this Commentary article we have attempted to answer the above-mentioned questions by reviewing the current knowledge on the molecular mechanisms controlling and sustaining the reaction of astroglia to injury and its stem cell-like properties. Moreover, the cellular/molecular mechanisms supporting the detrimental or beneficial features of astrogliosis have been scrutinized to gain insights on possible pharmacological approaches to enhance astrocyte neuroprotective activities.     

成年大鼠脊髓损伤后反应性星形胶质细胞增生和巢蛋白表达的相关性及意义

目的 探讨脊髓损伤后反应性星形胶质细胞的增生和巢蛋白的表达情况,并观察其细胞类型及相关性.方法 利用动脉瘤夹压迫法建立成年大鼠脊髓损伤动物模型.利用BBB评分标准表、免疫组化、免疫荧光双标法与LeicaQ500IW图像处理系统分析和显示脊髓损伤后不同时期(1、3、5 d,1、2、4、6、8、w)、不同部位脊髓损伤的程度以及巢蛋白与胶质酸性纤维蛋白(GFAP)的表达变化及细胞类型.结果 BBB评分结果显示术后所有实验动物双后肢(HL)评分低至0~1min,随后逐渐上升,1～2周内恢复的幅度较大,以后恢复较缓慢,较正常对照组具统计学意义(P<0.05).甲苯胺蓝染色可见损伤区有核固缩、胞浆溶解、尼氏体模糊且染色较深的神经元.随着动物存活时间延长,损伤范围逐渐扩大,约1周后损伤范同不再扩大.正常对照组脊髓神经元胞体和突起轮廓清晰.免疫荧光双标染色及图像处理系统分析结果显示脊髓伤24h后可诱导损伤及邻近区域巢蛋白和GFAP高度表达,中央管周围室管膜区几乎均为巢蛋白/GFAP-细胞群.室管膜区以外的灰质和白质中几乎全是巢蛋白/GFAP 细胞群,3～7d逐渐达到高峰,之后逐渐减弱,在损伤后2周左右恢复至对照组水平(P<0.05).正常对照组在脊髓中央管室管膜区有少量巢蛋白/GFAP-细胞,在室管膜区以外的灰质和白质中偶可见染色强度较弱的巢蛋白/GFAP 共存细胞.结论 成年大鼠脊髓损伤可诱导巢蛋白和GFAP的表达.巢蛋白的表达和反应性星形胶质细胞增生呈正相关,且表达多相互共存;星形胶质细胞和神经干细胞对脊髓损伤都有反应,并可能参与中枢神经系统损伤修复.     

Estradiol stimulates progenitor cell division in the ventricular and subventricular zones of the embryonic neocortex

Two distinct populations of cerebral cortical progenitor cells that generate neurons during embryogenesis have been identified: radial glial cells and intermediate progenitor cells. Despite advances in our understanding of progenitor cell populations, we know relatively little about factors that regulate their proliferative behaviour. 17-beta-Estradiol (E2) is present in the adult and developing mammalian brain, and plays an important role in central nervous system processes such as neuronal differentiation, survival and plasticity. E2 also stimulates neurogenesis in the adult dentate gyrus. We examined the role of E2 during embryonic cortical neurogenesis through immunohistochemistry, in situ hybridization, functional enzyme assay, organotypic culture and in utero administration of estradiol-blocking agents in mice. We show that aromatase, the E2 synthesizing enzyme, is present in the embryonic neocortex, that estrogen receptor-alpha is present in progenitor cells during cortical neurogenesis, that in vitro E2 administration rapidly promotes proliferation, and that in utero blockade of estrogen receptors decreases proliferation of embryonic cortical progenitor cells. Furthermore, the E2 inhibitor alpha-fetoprotein is expressed at high levels by radial glial cells but at lower levels by intermediate progenitor cells, suggesting that E2 differentially influences the proliferation of these cortical progenitor cell types. These findings demonstrate a new functional role for E2 as a proliferative agent during critical stages of cerebral cortex development.     

Transplanted glioma cells migrate and proliferate on host brain vasculature: a dynamic analysis

Glioma cells have a remarkable capacity to infiltrate the brain and migrate long distances from the tumor, making complete surgical resection impossible. Yet, little is known about how glioma cells interact with the complex microenvironment of the brain. To investigate the patterns and dynamics of glioma cell infiltration and migration, we stereotactically injected eGFP and DsRed-2 labeled rat C6 glioma cells into neonatal rat forebrains and used time-lapse microscopy to observe glioma cell migration and proliferation in slice cultures generated from these brains. In this model, glioma cells extensively infiltrated the brain by migrating along the abluminal surface of blood vessels. Glioma cells intercalated their processes between the endothelial cells and the perivascular astrocyte end feet, but did not invade into the blood vessel lumen. Dynamic analysis revealed notable similarities between the migratory behavior of glioma cells and that previously observed for glial progenitor cells. Glioma cells had a characteristic leading process and migrated in a saltatory fashion, with bursts of migration separated by periods of immobility, and maximum speeds of over 100 microm/h. Migrating glioma cells proliferated en route, pausing for as short as an hour to divide before the daughter cells resumed migrating. Remarkably, the majority of glioma cell divisions took place at or near vascular branch points, suggesting that mitosis is triggered by local environmental cues. This study provides the first dynamic analysis of glioma cell infiltration in living brain tissue and reveals that the migration and proliferation of transplanted glioma cells is directed by interactions with host brain vasculature.     

两种体外培养星形胶质细胞的比较

目的 对2种常用培养方法得到的星形胶质细胞进行生物学功能上的比较分析。方法 用啮齿类新生儿大脑星形胶质细胞分离培养方法培养原代星形胶质细胞,用胎牛血清分化神经干细胞制备分化星形胶质细胞。显微镜下观察2种星形胶质细胞的细胞形态并用免疫荧光染色检测星形胶质细胞特异性标志物胶质纤维酸性蛋白(GFAP)的表达量,10种细胞因子处理2种细胞后检测GFAP等的变化,用表达谱芯片检测GFAP、谷氨酸合成酶(GS)、转运蛋白(xCT)、神经调节蛋白-1(NRG)、谷氨酸受体蛋白(NMDA)、脂蛋白脂肪酶(LPL)等基因的表达并进行比较。结果 2种星形胶质细胞形态和GFAP表达量不同,表达谱芯片检测结果显示原代星形胶质细胞GFAP、GS、xCT、NRG、NMDA、LPL基因的表达均显著高于分化星形胶质细胞。10种细胞因子的处理均不能使原代星形胶质细胞GFAP表达升高,但转化生长因子-β(TGF-β)可以使分化的星形胶质细胞GFAP表达升高。结论 与分化的星形胶质细胞相比,原代培养的星形胶质细胞更类似于反应态星形胶质细胞,而TGF-β可促进分化细胞向反应态细胞过渡。     

一种肠肌间神经丛胶质细胞无血清原代培养法的建立

目的 肠胶质细胞（enteric glial cell,EGC）在肠道多种生理及病理过程的调节中发挥重要作用,为了深入研究EGC的功能,排除在体及组织水平中神经体液因素的影响,获得一种简便高效的原代EGC分离培养方法非常必要。方法 取小鼠结肠纵行肌-肌间神经丛组织,经消化分离肠胶质细胞,采用含血清和无血清培养基交替培养法去除成纤维细胞后常规传代;运用细胞免疫荧光染色法检测原代EGC纯度;运用胞内钙成像技术实时观察EGC内Ca²⁺浓度（[Ca²⁺]i）变化以检测细胞活性。结果 每次实验获得的EGC至传代前细胞量可达约6.8×10⁵;无血清培养后成纤维细胞污染显著降低;胶质纤维酸性蛋白（glial fibrillary acidic protein,GFAP）阳性的EGC占细胞总数的98.44%±1.07%,钙结合蛋白S100β阳性EGC占93.61%±3.16%,GFAP与S100β双阳性EGC占93.09%±2.99%;96.00%±4.00%的细胞在ATP诱导下发生胞内钙瞬变。结论 结合无血清培养可有效去除成纤维细胞污染,得到纯度较高、活性较好的肌间神经丛EGC。